U.S. patent number 5,009,185 [Application Number 07/350,106] was granted by the patent office on 1991-04-23 for apparatus for applying a controlled amount of reagent to a microscope slide or the like.
This patent grant is currently assigned to Wescor, Inc.. Invention is credited to Wayne K. Barlow, Gary D. Bradshaw, Barry O. Stokes.
United States Patent |
5,009,185 |
Stokes , et al. |
April 23, 1991 |
Apparatus for applying a controlled amount of reagent to a
microscope slide or the like
Abstract
Apparatus for applying a controlled amount of reagent to a
microscope slide or the like includes a spray chamber and a
carousel adapted to hold a plurality of slides and to be mounted
for rotation in the spray chamber. Nozzles are mounted to spray
reagent onto the slides in the carousel as they rotate by the
nozzles. Accurate control of the time during which the reagent is
sprayed from the nozzles is obtained by placing a control valve in
the flow line between a reagent pump and the nozzle and operating
the valve to control flow of reagent. Control circuitry coordinates
operation of the pumps, valves, and rotation of the carousel to
accurately cause spray of reagent onto the slides.
Inventors: |
Stokes; Barry O. (Logan,
UT), Bradshaw; Gary D. (Wellsville, UT), Barlow; Wayne
K. (Providence, UT) |
Assignee: |
Wescor, Inc. (Logan,
UT)
|
Family
ID: |
23375270 |
Appl.
No.: |
07/350,106 |
Filed: |
May 9, 1989 |
Current U.S.
Class: |
118/52; 118/314;
118/320; 118/56 |
Current CPC
Class: |
G01N
1/312 (20130101); G01N 2001/2846 (20130101); G01N
2001/317 (20130101) |
Current International
Class: |
G01N
1/30 (20060101); G01N 1/31 (20060101); G01N
1/28 (20060101); B05C 011/02 () |
Field of
Search: |
;118/56,314,320,52
;427/2 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Nozick; Bernard
Attorney, Agent or Firm: Mallinckrodt & Mallinckrodt
Claims
What is claimed is:
1. Apparatus for applying a controlled amount of reagent to a
microscope slide, comprising a spray chamber; carousel means for
holding a plurality of slides to be sprayed and rotatably mounted
in the spray chamber; nozzle means located in the spray chamber
adjacent a portion of the carousel means for directing spray of
reagent toward the slides held in the portion of the carousel means
adjacent the nozzle means; means for rotating the carousel means at
predetermined speeds in the spray chamber so as to move the slides
mounted in the carousel means past the nozzles means; reagent
reservoir means for storing a supply of reagent; pump means for
pumping reagent from said reservoir means to said nozzle means;
flow line means extending between said pump means and said nozzle
means through which reagent can flow between said pump means and
nozzle means; control valve means located in said flow line between
said pump means and said nozzle means to control flow of reagent
between said pump means and said nozzle means, the portion of said
flow line extending between said valve means and said nozzle means
being configured to eliminate pressure that would cause continued
flow of reagent from the flow line through the nozzle after closing
of the control valve whereby spray of reagent into the spray
chamber stops substantially immediately upon closing of the control
valve; means to determine and monitor the angular position of the
carousel means in the spray chamber; and control means responsive
to the means to determine and monitor the angular position of the
carousel means to coordinate operation of said pump means, valve
means, and carousel means to accurately cause spray of reagent
during predetermined known passage of the carousel means past the
nozzle means whereby spray of reagent onto the slides is accurately
controlled as the slides pass the nozzle means.
2. Apparatus for applying a controlled amount of reagent to a
microscope slide according to claim 1, wherein the control means
includes means for entering the number of slides in the carousel to
be stained when the number of slides in the carousel is less than
the maximum number the carousel can hold and wherein the control
means will coordinate operation of the pump means, valve means, and
carousel means to spray reagent only during the time a slide is in
position to receive the sprayed reagent.
3. Apparatus for applying a controlled amount of reagent to a
microscope slide according to claim 1, wherein all areas of the
flow line between the control valve and the nozzle are filled with
fluid during spraying and are nonexpandable under spraying
pressures.
4. Apparatus for applying a controlled amount of reagent to a
microscope slide according to claim 3, wherein the control valve is
located adjacent the nozzle to minimize the length of the flow line
between the valve and nozzle.
5. Apparatus for applying a controlled amount of reagent to a
microscope slide according to claim 1, wherein vent means is
provided to vent the flow line between the control valve and the
nozzle immediately upon closing of the control valve to stop fluid
flow to thereby relieve any pressure built up in the flow line
during spraying that would otherwise cause continuaton of spray
after closure of the control valve.
6. Apparatus for applying a controlled amount of reagent to a
microscope slide according to claim 5, additionally including a
check valve in the nozzle to prevent backflow of fluid from the
nozzle during venting of the flow line.
7. Apparatus for applying a controlled amount of reagent to a
microscope slide according to claim 5, wherein the control valve
includes a normally closed inlet port which is connected to the
output of the pump and which opens upon activation of the valve and
a normally open inlet port and wherein the vent means includes the
normally open inlet port connected to a venting source.
8. Apparatus for applying a controlled amount of reagent to a
microscope slide according to claim 7, wherein the venting source
is the fluid reservoir.
Description
BACKGROUND OF THE INVENTION
1. Field
The invention is in the field of automated equipment for staining
specimens on glass slides.
2. State of the Art
There are currently many medical diagnostic tests performed by
applying a biological material, such as blood, pus, urine or a
bacterial culture, on a glass slide, such as a microscope slide, by
smearing the material onto the slide, and then evaluating the
material applied to the slide. In most instances, the smear will be
treated with some type of reagent, such as a stain, to bring out or
make visible various features of the smear not otherwise visible.
In most cases the smear will also be fixed to the slide prior to or
concurrently with staining.
It is currently normal practice with hematology smears, i.e. blood
smears, to place a smear on a slide and allow the smear to dry. The
smear is then fixed on the slide by wetting the smear with
anhydrous methanol and allowing the methanol to evaporate or dry.
Dye is then applied to the smear to bring out desired
characteristics of the smear and the smear is then examined. While
the staining intensity achieved depends on the time and the amount
of stain applied, considerable variation in those variables still
yield satisfactory results.
In addition to blood smear staining, it is common to observe
bacteria in specimens and products as an indication of infection or
contamination. Material to be observed is usually applied to a
microscope slide, usually by smearing the material on the slide,
fixed, and stained using the well known gram staining procedure.
The gram staining consists of saturation of the smear with crystal
violet followed by a thorough water wash, and then saturation with
iodine followed by a thorough water wash. These steps are
non-critical and as long as enough crystal violet and iodine are
used to saturate the smear, any extra amount used does not affect
the results obtained. The critical step in gram staining is
decolorization. This is done by applying an organic solvent to
selectively remove the crystal violet-iodine complex from the gram
negative, but not the gram positive organisms. A counterstain
containing safranin is then applied to stain the gram negative
organisms red.
The rate of removal of the complex is species specific. If too
little decolorizer is added the gram negative organisms (red) will
appear gram positive (blue). If too much is added, the gram
positive organisms will appear gram negative. Reliable
decolorization can only be achieved where the amount of solvent
applied to the smear is accurately controlled.
U.S. Pat. Nos. 4,004,550 and 4,089,989 show an automated slide
stainer which automatically applies stain to slides mounted on a
carousel for rotation in a staining chamber. A commercial version
of the stainer shown in such patents is manufactured by Wescor,
Inc., Logan, Utah and sold under the trademark Aerospray.
In the commercial version of the stainer shown in the above cited
patents, slides to be stained are placed in a slide holding
carousel which is rotated by a motor, as shown in the patent.
During rotation of the carousel, the slides are sprayed with
various reagents as they move past a spray nozzle, again as shown
in the patents. However, it is impractical to use aerosol cans, as
shown in the patents, for spraying the various reagents. Rather,
pumps are provided for each of the reagents to pump the reagents
from their container or other reservoir through a check valve to
prevent back flow and through a nozzle which atomizes the reagent
and sprays it onto the slides in the spray chamber. In order to
spray a reagent onto the slide, the pump for the appropriate
reagent is energized. The reagent is then sprayed into the chamber
and onto the slides during the time its pump is energized. The
pumps are timed and controlled to pump the proper reagents into the
spray chamber at the proper times and for the proper length of
time. With such system, however, the spray continues to flow into
the chamber for 1 to 1.5 seconds during wind-down of the pump and
depressurization of the delivery line after power to the pump is
cutoff. This prevents highly accurate application of a reagent to
the slides. If the slide carousel rotates at 30 RPM, an additional
one second of spray application during wind down of the pump will
give half of the slides in the carousel an extra application of
reagent. Furthermore, the application of reagent to the slides is
not uniform as the pump looses pressure. This means that some of
the slides will receive an extra application of reagent and some of
the slides will receive uneven dribble. In the case of hematology
staining, as indicated above, this unevenness does not seem to be a
significant problem. The staining approaches a saturation point and
differences in staining due to this uneven application are not
detectable. However, in gram staining, the application of the
decolorization solvent must be accurately controlled and the extra
application of such solvent to some of the slides and not others
affects the accuracy and reproducibility of the gram staining
results. Thus, the currently commercially available automated
staining equipment made according to the referenced patents cannot
be used for optimal gram staining.
In addition to the problem encountered if such equipment is used
for gram staining, the additional stain used during wind down of
the pump wastes the various reagents used. Further, because of the
wind down time, it has not been practical to try to program the
equipment for use with less than a full load of slides. Thus, the
equipment is set to provide full pressure spray for the appropriate
number of full revolutions of the carousel, regardless of how many
slides are actually in the carousel. If the carousel is built to
hold twelve slides, and only one or two slides are to be treated,
the equipment will still spray enough reagent into the spray
chamber to treat twelve slides, thus additionally wasting
reagent.
SUMMARY OF THE INVENTION
According to the invention, apparatus for applying a controlled
amount of reagent to a slide or the like having a specimen thereon
includes a spray chamber and carousel means adapted to hold a
plurality of slides and to be mounted for rotation in the spray
chamber. Means is provided for rotating the carousel means in the
spray chamber and nozzle means are mounted in the spray chamber for
directing spray of reagent toward the carousel means. Reagent to be
applied to the slides is stored in a reagent reservoir and pump
means pumps the reagent from the reservoir through a flow line to
the nozzle when it is desired to spray the reagent on the slides.
Accurate control of the time during which the reagent is sprayed
from the nozzle is obtained by placing a remotely operated valve in
the flow line between the pump and the nozzle, energizing the pump
in anticipation of providing fluid to the nozzle, and operating the
valve to control the flow of fluid to the nozzle during the time
the pump is energized. This also involves either substantially
eliminating any compressible material, such as air pockets, or
expandable tubing in the line between the valve and the nozzle
which would cause continued spray of reagent through the nozzle
after closing of the valve, or venting the flow line between the
valve and nozzle immediately upon closing the valve to release any
pressure in the flow line caused by air pockets or expandable
material therein to prevent continued spray of reagent after the
valve is closed. Control means, such as a microprocessor and
related circuitry, coordinates operation of the pump, valve, and
carousel to accurately cause spray of reagent during known passage
of the carousel past the nozzle.
In one embodiment of the invention, the valve is located in the
flow line immediately adjacent the nozzle to substantially reduce
the volume of the flow line between the valve and nozzle thereby
eliminating excess spray from the nozzle. When the valve closes,
the spray stops.
In a second embodiment of the invention, substantially all air
space and resilient components which can expand under pressure are
eliminated from the flow line between the valve and the nozzle so
that pressure build up in the line is substantially eliminated and
flow of reagent through the line to the nozzle stops upon closing
of the valve.
In a third embodiment of the invention, the valve is connected so
that when it closes the flow line to the pump, it opens a vent in
the line between the valve and the nozzle to immediately equalize
the pressure in that line and cause the flow of reagent to the
nozzle to immediately cease. A check valve is included adjacent the
nozzle to prevent draining of reagent from the line between the
valve and nozzle so that upon connecting the line again to the
pump, spray through the nozzle occurs substantially
immediately.
With each of the embodiments indicated, the flow line between the
valve and nozzle, and the nozzle itself, is of sufficiently small
diameter to hold reagent in the line and keep it from running out
of the line through the nozzle.
THE DRAWINGS
In the accompanying drawings, which show the best mode presently
contemplated for carrying out the invention:
FIG. 1 is a perspective view of an apparatus of the invention;
FIG. 2, a vertical section taken on the line 2--2 of FIG. 1 showing
some of the internal components schematically;
FIG. 3, a flow diagram showing flow of reagent in the
apparatus;
FIG. 4, a longitudinal vertical section through a nozzle showing a
prior art nozzle configuration;
FIG. 5, a longitudinal vertical section similar to that of FIG. 4,
but showing a check valve of the invention therein;
FIG. 6, a longitudinal section similar to that of FIG. 4, but
showing an inset of the invention therein;
FIG. 7, a flow diagram similar to that of FIG. 3, but only showing
the left hand side thereof and showing a slightly different
embodiment;
FIG. 8, a flow diagram similar to that of FIG. 7, but showing the
valves in different order;
FIG. 9, a longitudinal section through another embodiment of a
nozzle of the invention; and
FIG. 10, a block diagram of a control system of the invention.
DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENT
An automatic slide stainer is shown in FIGS. 1 and 2 and includes
an outer housing 10 which encloses the operating components of the
equipment and has a control panel 11 thereon easily accessable to a
user of the equipment. A top cover 12 is hingedly mounted on top of
housing 10 as at 13 and opens to expose a spray chamber 14. Spray
nozzles 15, 16, 17, 18, and 19 are arranged about a portion of the
outside wall 20 of spray chamber 14.
A carousel 21 is adapted to fit into spray chamber 14 over a
receiving hub 22a secured to the end of shaft 22 and is adapted to
be rotated in the spray chamber 14 by shaft 22. Carousel 21 has a
central hub portion 23, a bottom portion 24 extending outwardly
from hub 23, and upstanding slide end holders 25 extending from
bottom portion 24 and preferably spaced evenly about the
circumference thereof. Hub 23 and slide end holders 25 have slots
26 therein to receive the ends of microscope slides 27 which can be
placed to extend between the hub 23 and a slide end holder 25. It
is preferred that carousel 21 be provided with twelve slide end
holders 25 so that up to twelve individual slides can be mounted in
the carousel, preferably in balanced configuration about the
carousel. The bottom portion of the carousel forms a collecting
area for reagent which can run out of the collecting area through
circumferentially placed slots 28. A carousel cover 30 fits over
the top of the carousel 21 and is held in place by a bayonet
latching mechanism 31 which engages with a receiving hole 32 in
carousel hub 23 and has an enlarged head 33 which allows the
engaging mechanism to be easily secured or removed from the hub and
serves as a handle for holding and loading the carousel into spray
chamber 14.
Shaft 22 extends through a seal 35 in spray chamber bottom 36 and
is coupled for rotation to motor 37, shown schematically in FIG. 2.
Thus, operation of motor 37 causes rotation of shaft 22 and
receiving hub 22a secured to the end thereof which, in turn, causes
rotation of carousel 21 when mounted thereon in spray chamber 14.
Carousel hub 23 and receiving hub 22a preferably include some type
of indexing, such as mating flattened sides shown at 23a so that
carousel 21 is always positioned in known manner with regard to
shaft 22.
In operation of the apparatus, the motor is operated to rotate the
carousel at a known rate and reagent to be applied to the slides in
the carousel is sprayed through one or more of the nozzles into the
spray chamber. As the carousel rotates, with each rotation of the
carousel, each of the slides pass by the nozzles and the reagent
sprayed from the nozzles is applied to the slides as they pass by.
By controlling the speed at which the carousel rotates and timing
the spray of reagent from the nozzles, a desired amount of reagent
can be applied to the slide. While five nozzles have been shown in
the spray chamber of FIG. 1, and this is preferred for use in gram
staining where three of the nozzles are used for reagent, each
nozzle supplying a different reagent at a different time, and two
of the nozzles are used to supply a wash fluid, any number of
nozzles may be used and a single nozzle could be used to supply
different reagents at different times.
As shown schematically in FIG. 2, nozzle 15 is mounted in a nozzle
holder 40 with a tubing connector 41 extending therefrom. Tubing 42
extends from connector 41 to a remotely operated wash valve 43.
Tubing 44 extends from wash valve 43 to a remotely operated control
valve 45. Tubing 46 extends from control valve 45 to the output
side of pump 47. The inlet side of pump 47 is connected through
tubing 48 to a source of reagent, such as a bottle 49 containing a
reagent 50.
In the equipment that has been manufactured and sold under U.S.
Pat. Nos. 4,004,550 and 4,089,989 as mentioned in the State of the
Art Section of this application, tubing extends directly from pump
47 to normally open wash valve 43 with tubing then extending to
nozzle holder hose connector 41. Control valve 45 has not been used
with such equipment. Without the control valve, when it is desired
to spray reagent onto the slides in the carousel rotating in the
spray chamber, pump 47 is energized by a timing and control means
51 and reagent is drawn by pump 47 from container 49 and supplied
through the open wash valve directly to nozzle 15. This results in
the problems previously mentioned that spray continues for a short
time after the pump is deenergized while the pump winds down.
With the arrangement of the invention where the spray of the
reagent into the spray chamber 14 has to be accurately controlled,
the remotely controlled control valve 45 is placed in the flow line
between the pump and the nozzle to more accurately control the flow
of reagent from the pump to the nozzle.
In the particular embodiment of FIGS. 2 and 3, control valve 45 is
provided between the pump and wash valve 43. Valve 45 controls flow
of reagent from the pump to the nozzle. Wash valve 43 is provided
to allow flow of a wash solution, such as water, through the nozzle
prior to ceasing operation of the apparatus for a period of time,
such as overnight. Operation of valve 43 will be explained in more
detail later. With gram staining, only the flow of the
decolorization and counterstaining reagent is critical to control
so it is only necessary to provide a flow control valve in the line
for one of the nozzles. The remaining nozzles do not require
critical control of the reagent pumped to the nozzles so flow
control valves are not necessary in the other lines.
FIG. 3 shows a flow diagram for the five nozzles provided in the
apparatus of FIG. 1. The operation of the apparatus will be
discribed specifically for gram staining.
Slides having bacterial specimens thereon are placed in the
carousel and the carousel is positioned on receiving hub 22a in
spray chamber 14. Prior to placing the slides in the carousel, the
bacterial specimens generally will have been fixed to the slides in
normal manner, such as by heating or by an application of methanol.
Top 12 is closed over the spray chamber. The operator then selects
the desired operations and starts operation of the apparatus by
means of controls on control panel 11.
Upon the initiation of operation of the apparatus, control means 51
energizes motor 37 to cause rotation of the carousel. The carousel
is operated at a known rate of rotation, for example, thirty RPM,
for the spraying operation. The first step in gram staining is
saturation of the smear with crystal violet. Crystal violet is
provided in one of the reagent reservoirs, such as bottle 60, FIGS.
1 and 3. Pump 61 is energized to draw the crystal violet from
reservoir 60 to pump 61 through tubing 62. The crystal violet is
pumped through tubing 63 to the normally open inlet of wash valve
64, through open valve 64, and tubing 65, to nozzle 16. The control
means 51 energizes pump 61 to start flow of crystal violet and
continues operation of pump 61 for a set number of full revolution
of the carousel to saturate the specimens on the slides with
crystal violet. It has been found that a ten second spray time (5
full revolution of the carousel) is generally satisfactory. After
the set number of revolutions, pump 61 is deenergized and spray
continues during wind down of the pump. However, since this spray
is a saturation operation, the extra spray during wind down of the
pump does not affect the results.
After application of the crystal violet, it has been found
beneficial to increase the speed of rotation of the carousel to
between 500 and 1000 RPM for three seconds to remove the excess
crystal violet reagent from the slides. The rotation speed is then
returned to thirty RPM and a water wash is provided for ten to
sixteen seconds. It usually requires about sixteen seconds to slow
the carousel down to thirty RPM from the high speed spin cycle and
be sure that the speed has stabilized at the lower speed.
For the water wash, water is provided in bottle 66 which is drawn
through tubing 67 to the inlet of pump 68. Pump 68 then pumps the
water through tubing 69 to manifold 70, through tubing 71 and 72 to
nozzles 18 and 19. Nozzle 18 is mounted in spray chamber 14
similarly to nozzles 15, 16, and 17 to provide spray to the front
surface of the slides, i.e. the surface with the specimens thereon,
as they pass the nozzle. Nozzle 19, however, is arranged to spray
the back side of the slides as they pass.
After saturation with crystal violet and the subsequent water wash,
the next step is to saturate the specimen with iodine. The iodine
is supplied in bottle 74. Pump 75 is energized to draw iodine from
bottle 74 through tubing 76 to the inlet of pump 75. The iodine
reagent then flows from the output of pump 75 through tubing 77,
normally open wash valve 78, tubing 79, to nozzle 17. The pump is
operated by control means 51 similarly to pump 64 to saturate the
smear with iodine. Ten seconds of iodine spray has been found
satisfactory.
After application of the iodine, the speed of the carousel is
increased for a three second high speed cycle (500-1000 RPM),
followed by a return to the thirty RPM speed and a twelve to
sixteen second water wash.
After saturation with iodine and the subsequent water wash, the
next steps in gram staining are the critical steps of
decolorization and counterstaining. With the apparatus of the
invention, the decolorization solvent and the counterstain safranin
are mixed together and applied together in a single spray step.
This step requires the accurate application of the organic solvent
containing the red counterstain safranin to the slides. The
decolorization solvent and counterstain solution 50, FIG. 2, is
supplied in bottle 49. Pump 47 draws the counterstain solvent from
bottle 49 through tubing 48 to the pump and then pumps it through
tubing 46 to normally closed valve 45. In order to provide accurate
control of the spray of the counterstain solvent, pump 47 is
energized by control means 51 one second prior to the time spraying
is to start. This allows the pressure in pump 47 and line 46 from
the pump to build up and stabilize. When spraying is to start,
control means 51 causes operation of valve 45 to open the normally
closed inlet of the valve so that counterstain solvent flows
through the valve, through tubing 44, through normally open valve
43, and through tubing 42 to nozzle 15. Tubing 42 and 44 is
arranged so that it remains full of fluid between operations so
with the pressure from the pump stabilized prior to opening valve
45, spray of the solvent through nozzle 15 begins immediately upon
opening valve 45. Upon initially opening valve 45, control means 51
keeps valve 45 open to allow two complete revolutions of the
carousel. Thus, each slide in the carousel passes by the spray
nozzle twice. With a rotation rate of thirty RPM, each complete
rotation takes two seconds so the initial spray is timed to
continue for four seconds. At the end of four seconds, control
means 51 causes valve 45 to close. This immediately causes the
spray through nozzle 15 to cease.
Since the counterstain solvent must be absorbed into the specimen
to work and decolorization and counterstaining does not occur
immediately, it is usually necessary to apply the counterstain
solvent, allow some time for it to be absorbed into the specimens,
apply some more counterstain solvent, allow time for it to be
absorbed, and continue the intermittent application of counterstain
solvent with absorption periods until the desired decolorization
and counterstaining takes place. Because the desired amount of
decolorization and counterstaining varies from user to user and
because the thickness of the specimens and thus the amount of
counterstain solvent required for a given specimen or batch of
specimens will vary, it will usually be advantageous to allow the
user to determine the number of applications of counterstain to be
applied and to enter this number via the control panel keys prior
to start of operation of the machine. Usually, it will be required
to provide several additional applications of the counterstain
solvent so the control means 51 will cause operation of the spray
every four seconds (or other set or selectable time period) for the
number of applications selected by the user. Thus, after the stop
of the initial spray application, control means 51 will keep pump
47 energized. At four seconds after stop of the initial spray, i.e.
after two complete revolutions of the carousel to allow the initial
application of counterstain solvent to penetrate and decolorize and
counterstain the specimen, control means 51 causes valve 45 to open
to start spray again through nozzle 15. Valve 45 remains open for
two seconds (the additional applications of counterstain solvent
will usually be timed for one complete revolution of the carousel
rather than the two revolutions of the initial application),
providing an additional application of counterstain solvent to each
side. At the end of the two second period, valve 45 is closed to
immediately stop the spray. Additional applications of solvent are
repeated every four seconds until the selected number of
applications have been made. In some cases, it has been found
desirable to increase the time between the later cycles to six or
eight seconds rather than four seconds to allow additional time for
the decolorization and counterstain to be absorbed into the
specimen. Usually no more than six applications of counterstain
solvent will be desired.
After the desired number of applications of counterstain solvent, a
water wash is applied for six seconds to stop decolorization, and
then carousel speed is increased for a three second period. The
carousel is then slowed to the thirty RPM rate for an eight to
fourteen second water wash, followed by a thirty second high speed
cycle to dry the slides. The slides are then removed and examined.
The apparatus is then ready to stain another batch of slides.
If specimens on the slides to be stained have not been fixed to the
slides prior to the slides being loaded into the carousel, the
specimens can be fixed to the slides by an initial application of
the counterstain solvent. In such instance, in order to fix the
specimens, upon start up of the apparatus, a four second spray of
counterstain solvent is applied to the specimens in the same manner
by pump 47, control valve 45 and nozzle 15, as described above. The
carousel continues spinning at thirty RPM for twenty seconds to
allow fixing of the specimen to take place. This is followed by a
three second high speed spin and then a ten second water wash. This
is followed by the normal staining cycle described above starting
with the application of the crystal violet reagent. However, more
crystal violet reagent is used to compensate for the decolorizer
that was initially applied to the specimens during fixing. Thus,
crystal violet is applied for fifteen seconds followed by a fifteen
second wait and a three second high speed spin and then an eight
second application of crystal violet. This is followed by a fifteen
second wash and then a three second high speed spin. The iodine is
then applied and the remaining steps are as described above.
When the apparatus is not going to be used for a period of time,
such as overnight, it is usually desireable to rinse all reagent
nozzles. In order to do this, pump 68 is energized to pump water to
manifold 70 and wash valves 43, 64, and 78 are operated to open the
normally closed inlet. This allows water to flow from manifold 70
through tubing 80 to valve 43, and through tubing 42 and nozzle 15
to rinse it. Similarly, water flows through tubing 81 to valve 64
and through tubing 65 and nozzle 16, and through tubing 82 to valve
78 and through tubing 79 and nozzle 17.
In order to ensure that the spraying during critical reagent
spraying through nozzle 15 starts immediately upon opening of
control valve 47 and stops immediately upon closing of control
valve 47, it is necessary to ensure that the flow line between the
valve and nozzle remain full of fluid between cycles and that any
compressable material, such as air pockets, or expandable tubing,
be eliminated from the line or that a pressure build up in the line
during spraying because of these factors is immediately released
upon close of the valve.
In the embodiment shown in FIGS. 2 and 3, valve 45 has normally
open and normally closed inputs. As previously described, the
output from pump 47 is connected by tubing 46 to the normally
closed input of valve 45. Thus, valve 45 must be opened to allow
flow of fluid from the pump to the nozzle. The normally open input
of valve 45 is connected through tubing 83 back to the supply line
48 from fluid supply bottle or reservoir 49 which is at
substantially atmospheric pressure. Thus, line 83 serves as a vent.
Immediately upon closing of valve 45 to stop the flow of fluid from
pump 47 to nozzle 15, the flow tubing between valve 45 and nozzle
15 is vented to atmospheric pressure through the normally open
input of valve 45 and tubing 83. This immeidiately provides relief
for any pressure built up in the line. With this relief, however,
it is necessary to provide a check valve in the line to stop back
flow of fluid from the nozzle which would empty the nozzle and flow
lines of fluid.
The normal nozzle used with the prior art apparatus discussed is
shown in FIG. 4 and includes the nozzle 15 threaded into nozzle
holder 40. Nozzle holder 40 is mounted in an opening in spray
chamber side wall 20 and is secured therein by a nut 84. An O-ring
85 seals the connection between the nozzle 15 and nozzle holder 40.
A threaded opening 86 accepts hose connector 41. Within the central
bore 87 of nozzle 15, against nozzle spray opening 88, is a spray
forming member 89. Spray forming member 89 is held in place by
threaded member 90 with central bore 91 therein. Radial openings 92
extend through member 90 toward its forward end. The forward
surface of spray forming member 89 which rests against the forward
closed end of nozzle bore 87 has grooves 93 therein for passage of
fluid to nozzle opening 88.
In use, fluid to be sprayed is introduced under pressure through
opening 86 in nozzle holder 40 and flows into larger opening 94 of
the nozzle holder 40 and into bore 87 of nozzle 15. The fluid flows
through central bore 91 in threaded member 90 through radial
openings 92 therein, around the outside of the forward ends of
members 90 and 91 and through passages 93 to nozzle outlet 88. This
prior art nozzle arrangement works well where spray control is not
critical, however, the extra area in the back of nozzle bore 87 and
in holder opening 94 generally will have air therein and during
spraying, the fluid will compress the air and partially fill the
space with fluid. In the actual equipment, this space in about 1 ml
and under normal operating conditions about one third to one half
of the space is filled with air. When spraying ceases, the air
trapped therein, which has been compressed, will expand and force
some of the fluid therein out through the nozzle. This produces an
undesireable afterspray.
FIG. 5 shows the same nozzle arrangement but includes a check valve
of the invention therein to prevent back flow of the fluid from the
nozzle and to eliminate the extra air and fluid space in the nozzle
holder and nozzle. The check valve includes a valve body 95 with a
threaded end to screw into threaded bore 87 of nozzle 15 and an
opposite end which fits into nozzle holder opening 94. A hollow
cylindrical insert 96 slides into check valve body 95 and has a
central shaft 97 extending from insert end 98. A series of openings
99 extend through end 98. A resilient disc 100 is positioned in
valve body 95 with its center against shaft 97. In operation,
pressurized fluid passes from nozzle holder opening 86, through
opening 101 in check valve body 95, around the outer edges of disc
100, which displace to allow fluid flow there around, through the
open areas of valve insert 96, and through openings 99 in insert
end 98 into bore 91 in member 90. When flow of pressurized fluid
stops, any reverse flow causes disc 100 to seal against the inside
end of valve body 95 to seal opening 101 and prevent any reverse
flow.
An alternate embodiment is shown in FIGS. 6 and 7. In this
embodiment, valve 45 does not have a normally open input, or, the
normally open input is sealed. Thus, no venting of the fluid tubing
takes place. Steps must be taken to substantially eliminate any
sources of pressure in the line so that the spray will stop upon
closing of valve 45. Thus, the tubing used should not stretch under
the pressures used and the air and fluid areas in the nozzle holder
and nozzle should be blocked. For this purpose, it has been found
that a length of silicone tubing 110, FIG. 6, can be placed between
the nozzle member and the base of nozzle holder opeing 94. The
silicone tube compresses to form a seal against nozzle holder 40
and nozzle member 92 and closes the air and fluid space otherwise
available. It has been found that with this arrangement, spray
stops upon closing of the valve 45. Since valve 45 does not vent
upon closing, there is no need for a check valve at the nozzle.
Closed valve 45 prevents any back flow of fluid from the nozzle.
With either embodiment, valve 45 can be located anywhere in the
line between pump 47 and nozzle 15. It can be on either side of
valve 43.
FIGS. 8 and 9 show a further alternate embodiment. In the
embodiment of FIG. 8, valve 45 again does not have a normally open
inlet port or the normally open inlet port is sealed. Again, there
is no venting. In this embodiment, valve 45 is shown between valve
43 and nozzle 15 and is preferably located as close to nozzle 15 as
possible to reduce the amount of flow line between the valve and
nozzle.
FIG. 9 shows an arrangement for mounting valve 45 adjacent nozzle
15. In this embodiment, nozzle 15 is threaded onto a nipple 120.
Nipple 120 passes through spray chamber side wall 20 and is held in
place by nuts 121 and 122 threaded onto nipple 120 and tightened
against the opposite sides of wall 20. This mounts nozzle 15 in the
spray chamber. It should also be noted that the nipple 120 provides
a small flow passage into the valve and does not leave any air
space at the back of the valve. Valve 45, shown only partially in
FIG. 9, is mounted directly to nipple 120, thus providing a very
short flow passage between the valve and nozzle 15. In this
embodiment, valve 45 also acts as the check valve to prevent back
flow of fluid from the nozzle.
While the carousel is configured to hold a plurality of slides, the
embodiment shown holds twelve slides, any number of slides up to
the full number of slides can be stained at any time. Thus, the
apparatus can be operated to stain a single slide which may be
placed in the carousel. However, when only a single slide is
stained, or any number of slides less than a full carousel are
stained, the apparatus as described above for gram staining, or the
prior art apparatus used for hematology staining, operates as if
the carousel was full. With ony several slides in the carousel,
such operation wastes reagents and in some cases it would be
desireable to control reagent spray so that the spray occurs only
during the time the slides in the carousel are passing by a nozzle.
Such controlled operation was difficult with the prior art
apparatus which merely activated the pump to start the spray since
the afterspray would usually continue through about an additional
half revolution of the carousel so the potential savings of reagent
by stopping the pump sooner for a partially loaded carousel than
for a fully loaded carousel was not significant. However, with the
control system of the present invention which allows the accurate
on-off control of the spray of reagent, in some cases it becomes
worthwhile to control the spray of such reagent for the specific
number of slides actually in the carousel. For such control, upon
the start of operation of the apparatus, the user indicates the
number of slides in the carousel and their positions via the
control panel. The control means then operates the control valve to
start the spray of reagent just prior to the slide passing the
nozzle and stops spray just after passage of the slide by the
nozzle.
The control means may be as simple or complex as desired and will
usually take the form of electronic timing and control circuitry to
operate and control the time of operation of the various pumps and
valves. For more complex control, the control circuitry may include
a microprocessor programmed to operate the apparatus in certain
ways in response to input signals from the control panel, or may be
custom programmed by the user via the control panel.
FIG. 10 is a block diagram of a basic microprocessor control means
for the invention. The control panel as shown in FIG. 1 comprises
three buttons 150-152 representing "clean", "stop", and "run"
functions, respectively, and six number push buttons 153 for
entering numbers. A display 154 displays instructions entered or
other information supplied by the microprocessor. The control panel
block in FIG. 10 provides information to the microprocessor or
receives information from the microprocessor to be displayed.
An encoder 155 is coupled to shaft 22 in motor 37, FIG. 2, and
shown by a block feeding into the microprocessor in FIG. 10. The
encoder provides signals to the microprocessor indicative of the
position and speed of rotation of shaft 22. Various encoders may be
used with an encoder that provides two separate outputs, one
providing 500 pulses per revolution and the other providing two
pulses per revolution, one pulse for each 180.degree. of rotation
of the shaft, has been found satisfactory. The encoder is used to
measure motor speed so speed can be accurately controlled by the
microprocessor, the basic function if individual slide staining
control is not provided, and to indicate position of the carousel
with respect to the nozzles if individual slide staining control is
provided. With the individual slide staining control, the 500
pulses per revolution is divided into twelve sets of pulses
representing twelve corridors or time periods representing each of
the individual slides in the carousel. Thus, the microprocessor can
keep track of the position of the individual slides and activate
the nozzles to spray the appropriate number of individual slides.
The pulses provided for each half revolution are used to keep track
of half or full revolutions and to correct for the fact that the
500 pulses do not evenly divide into the twelve corridors.
The microprocessor provides signals to the motor, the pumps, and
the valves to operate them at the appropriate times. The pumps and
valves are shown collectively by respective blocks in FIG. 10.
Thus, the block labeled "Pumps" may have four individual pumps as
shown in the embodiment described, or any other number of pumps
appropriate for a particular embodiment.
While various microprocessors can be used, a Motorola 68HC11 has
been found satisfactory. The various interfaces for inputs and
outputs to the microprocessor are not shown, but would be obvious
to a person skilled in the art.
Various reagents may be used with the equipment. For the gram
staining described, it has been found that a counterstain solvent
made up of 60% isopropanol, 40% methanol, and 2.5 gram/liter
safrinin works well.
Also, while only the reagent line for the counterstain solvent has
been shown and described with the valve for accurate control of the
spray, similar control valves and nozzle and flow line
configurations can be on any of the other reagent lines or all of
the other reagent lines. Where the apparatus is adapted to control
the spray for less than a full carousel load of slides, i.e. to
control the spray so that reagent is sprayed only during the time a
slide is in position to receive the sprayed reagent, all reagent
lines will preferably have a control valve.
While the invention has been described in connection with gram
staining and certain modes of operation have been described in
detail, it should be realized that the various spray times, rate of
rotation of the carousel, and even the sequence of some of the
steps may be varied for the gram staining, and that various other
types of staining can be performed using the apparatus with varying
reagents and steps.
Whereas this invention is here illustrated and described with
specific reference to embodiments thereof presently contemplated as
the best mode of carrying out such invention in actual practice, it
is to be understood that various changes may be made in adapting
the invention to different embodiments without departing from the
broader inventive concepts disclosed herein and comprehended by the
claims that follow.
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